The N-terminal truncated pyroglutamate (pGlu) modified forms of beta-amyloid (A ), pGluA (3-40/42) (referred to as pGluA ), display high neurotoxicity, neurodegeneration and memory loss in Alzheimer's disease. Notably, pGluA is abundant in AD brains and is present at levels greater than A (1-40/42). The pGluA accelerates formation of A and pGluA oligomers that cause neurotoxic cell death and memory deficits in AD. The pGluA (3-40/42) peptides begin with N-terminal glutamate, the third amino acid of A (1-40/42). pGluA peptides are generated from A (1-40/42) by aminopeptidases that remove the N-terminal aspartate and alanine residues, followed by cyclization of the N-terminal glutamate. The aminopeptidases required to generate pGluA peptides have not yet been elucidated. Therefore, the goal of this project will be to identify the aminopeptidase mechanisms responsible for generating neurotoxic pGluA that participates with A in development of AD. Results can provide novel protease targets for Alzheimer's disease. The hypothesis of this project is that the aspartate aminopeptidase and alanine aminopeptidase enzymes generate A (3-40/42) which is converted to pGluA(3-40/42) by glutaminyl cyclase (QC). This hypothesis is supported by our preliminary data demonstrating the presence of aspartate and alanine aminopeptidase activities in secretory vesicles that contain pGluA (3-40/42) and A (1-40/42). This hypothesis will be assessed in three specific aims to (1) identify aspartate and alanine aminopeptidases in A -producing secretory vesicles that remove the N-terminal Asp and Ala residues from A (1-40/42), (2) evaluate identified aminopeptidases by gene silencing and expression for their roles in producing pGluA (3-40/42), and (3) evaluate inhibitors of these aminopeptidase activities to reduce production of pGluA (3- 40/42). This project will identify new aminopeptidase mechanisms for producing neurotoxic pGluA peptides. Results will also yield candidate inhibitors of these aminopeptidases for future investigation of aminopeptidase regulation in Alzheimer's disease.